1. Field of the Invention
This invention relates to a process for the production of pyruvic acid, where L-lactic acid and oxygen are reacted in an aqueous solution in the presence of a permeabilized whole cell catalyst which contains the enzymes glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15) and catalase (EC 1.11.1.6).
2. Description of the Related Art
Pyruvic acid has been prepared by the fermentation of various carbon sources (e.g., glucose, yeast extracts and peptone), but these methods usually produce pyruvic acid in low yields (based on added carbon source) and in relatively low concentrations as one component of a mixture of fermentation products. Separation and isolation of pyruvic acid from such complex fermentation broths are generally difficult and expensive to perform.
The preparation of pyruvic acid via the microbiological oxidation of optically pure D(-)-lactic acid has been described by Cooper (U.S. Pat. No. 4,900,668; Feb. 13, 1990). Although this process improves upon other fermentation routes by not utilizing the D-lactic acid as a carbon source to produce the cell mass necessary for the reaction, a growth medium containing a second carbon source (e.g., D(-)-mannitol and corn steep liquor) are required for both the production of cell mass as well as the fermentative conversion of D-lactic acid. Additionally, D-lactic acid is not as ubiquitous in nature, and is much more expensive to produce or purchase, than L(+)-lactic acid.
The conversion of L-lactic acid to pyruvic acid has been demonstrated using the enzyme L-lactate oxidase (L-lactate: oxygen oxidoreductase, non-decarboxylating, EC 1.1.3.2 ) as catalyst (B. A. Burdick and J. R. Schaeffer Biotech. Lett., Vol. 9, 253-258 (1987)). L-lactate oxidase (from Pediococcus) catalyzes the oxidation of L-lactate by oxygen to pyruvate and hydrogen peroxide: ##STR1## The L-lactate oxidase was co-immobilized with catalase (oxidase:catalase=1:281 (IU/IU)) to limit oxidation of pyruvate by by product hydrogen peroxide, which produces acetate and carbon dioxide. The oxidation of 0.049M solutions of L-lactate in 0.1M phosphate buffer at pH 7 resulted in yields of pyruvic acid (isolated as the 2,4-dinitrophenylhydrazone derivative) of from 0% to 47% (from 0.0M to 0.023M pyruvic acid); reusing the co-immobilized lactate oxidase/catalase catalyst in a second oxidation reaction resulted in significantly lower yields of pyruvic acid than were obtained in the first reaction.
Glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15), an enzyme commonly found in leafy green plants and mammalian cells, catalyzes the oxidation of glycolic acid to glyoxylic acid. This same enzyme also catalyzes the oxidation of L-lactic acid to pyruvic acid, with the concomitant production of hydrogen peroxide. C. O. Clagett, N. E. Tolbert and R. H. Burris, J. Biol. Chem., Vol. 178, 977-987 (1949) first reported an .alpha.-hydroxy acid oxidase, extracted from a variety of green leafy plants, which catalyzed the oxidation of glycolic acid, and was also specific for the L-isomer of lactic acid. The pH optimum for the oxidation of 80 mM dl-lactate was 7.6; no reaction products were identified or isolated. N. E. Tolbert et al., J. Biol. Chem., Vol. 181, 905-914 (1949) employed a purified .alpha.-hydroxy acid oxidase from tobacco leaves for the oxidation of ca. 113 mM dl-lactic acid in phosphate buffer at pH 8; an unreported quantity of pyruvic acid was isolated from the reaction as a 2,4-dinitrophenylhydrazone, and a significant amount of carbon dioxide was also produced, indicating that a significant amount of pyruvate had reacted with co-product hydrogen peroxide to produce acetate and carbon dioxide. K. E. Richardson and N. E. Tolbert, J. Biol. Chem., Vol. 236, 1280-1284 (1961) later reported that this .alpha.-hydroxy acid oxidase was more commonly referred to as glycolic acid oxidase (i.e., glycolate oxidase).
I. Zelitch and S. Ochoa, J. Biol. Chem., Vol. 201, 707-718 (1953) reported that glycolic acid oxidase catalyzes the oxidation of L-lactic acid by molecular oxygen to produce pyruvic acid and hydrogen peroxide, and that in the absence of catalase, the peroxide reacts non-enzymatically with pyruvate to form acetate, CO.sub.2, and water. Flavin mononucleotide (FMN) was identified as a required enzyme cofactor, and the addition of FMN to aqueous solutions of the enzyme greatly increased the stability of glycolic acid oxidase. The oxidation of 3.3 mM solutions of L-lactate in 50 mM phosphate buffer (pH 8.0) and in the presence of an excess of added catalase produced 3.2 mM pyruvic acid (determined colorimetrically); no product was isolated.
The oxidation of lactate to pyruvate has also been demonstrated using an L-.alpha.-hydroxy acid oxidase isolated from rat kidney (M. Blanchard et. al., J. Biol. Chem., Vol. 163, 137-144 (1946)). The oxidation of a 33 mM solution of lactate in 0.167M phosphate buffer at pH 8.0 and in the presence of added excess catalase produced pyruvate in 79% yield (isolated as the 2,4-dinitrophenylhydrazone derivative).
Additional references to the oxidation of lactic acid to pyruvic acid catalyzed by soluble enzymes include: J. C. Robinson et at., J. Biol. Chem., Vol. 237, 2001-2010 (1962 ) (hog renal cortex L-.alpha.-hydroxy acid oxidase), P. Urban et. al., Biochemistry, Vol. 27, 7365-7371 (1988) (rat kidney L-.alpha.-hydroxy acid oxidase), D. W. Fry and K. E. Richardson, Biochim. Biophys. Acta, Vol. 568, 135-144 (1979) (human liver glycolic acid oxidase), M. J. Emes and K. H. Erismann, Int. J. Biochem., Vol. 16, 1373-1378 (1984) (Lemna minor L. glycolate oxidase), H. S. Kim and J. D. Choi, Korean Biochem. J., Vol. 20, 350-356 (1987) (spinach glycolate oxidase).
Although the enzyme-catalyzed oxidation of L-lactic acid by oxygen is well-known, a high selectivity to pyruvic acid has only been demonstrated in one experiment (I. Zelitch and S. Ochoa) where the concentration of L-lactate was 3.3 mM; this low concentration of L-lactate limited the concentration of hydrogen peroxide formed, and in the presence of an excess of catalase, also limited the reaction of hydrogen peroxide with pyruvate to produce acetate and carbon dioxide. The recovery of such a low concentration of pyruvate (ca. 3.2 mM) from an aqueous reaction mixture is impractical for an economical manufacturing process.